178 TRANSCRIPT ABUNDANCE OF METHYLTRANSFERASES SPECIFIC FOR H3K9 DIFFER AT DISCRETE STAGES OF PORCINE OOCYTE AND CLEAVAGE STAGE EMBRYO DEVELOPMENT

2009 ◽  
Vol 21 (1) ◽  
pp. 188
Author(s):  
M. N. Biancardi ◽  
L. Magnani ◽  
C. M. Johnson ◽  
R. A. Cabot
Keyword(s):  
Germinal Vesicle ◽  
Transcript Abundance ◽  
Covalent Modification ◽  
Cleavage Stage ◽  
Transcript Levels ◽  
Vitro Fertilization ◽  
Multiple Comparison Test ◽  
Porcine Oocyte ◽  
Stage Embryo

Covalent modification of histone proteins plays a key role in transcriptional regulation. Methylation of the lysine residue 9 of histone protein 3 (H3K9) is globally remodeled during cleavage development. The aim of this study was to determine the relative transcript abundance of the 5 histone methyltransferases (HMTases) that have been shown to methylate H3K9, in porcine oocytes and cleavage stage embryos. We hypothesized that transcript levels for each of these HMTases (Suv39h1, Suv39h2, ESET, GLP, and G9a) would differ in their respective abundance at each developmental stage and that some would undergo dramatic changes in transcript abundance during development. To test this hypothesis, quantitative RT-PCR was performed on mRNA isolated from pools of 78–457 germinal vesicle (GV) and metaphase II (MII)-stage oocytes and 4-cell (4C) and blastocyst (BL)-stage embryos produced by either parthenogenetic activation (PA) or in vitro fertilization (IVF); each pool constituted an experimental replicate, and a minimum of 3 replicates were performed for each stage. Along with the 5 HMTases, transcripts for YWHAG were assayed and used as a normalizer. PCR data were quantified using Δ CT and 2ΔΔ CT methods; in the latter case, Δ CT values from the GV stage were used as the calibrator. All data were analyzed by ANOVA and Tukey’s multiple-comparison test. In GV oocytes, Suv39h2 transcripts were in the highest abundance, while ESET transcripts were in the lowest abundance, ESET transcripts being 1450-fold less abundant than Suv39h2 (P < 0.05). In MII oocytes, Suv39h2, GLP, and G9a transcripts were present in highest abundance and were not significantly different from each other; Suv39h1 transcripts were 18-fold less abundant, and ESET transcripts were 6650-fold less abundant than GLP transcripts (P < 0.05). ESET transcripts were present in the least abundance in 4C-PA embryos, 84-fold less than GLP (P < 0.05); no difference in transcript levels for Suv39h1, Suv39h2, GLP, and G9a were detected. ESET transcripts were not detectable 4C-IVF embryos. ESET transcripts were present in the least abundance in BL-PA embryos, 39-fold less abundant than GLP; there was no difference in transcript abundance between Suv39h1, Suv39h2, and G9a. In BL-IVF-stage embryos, ESET transcripts were in the lowest abundance, 97-fold less abundant than GLP (P < 0.05). Analysis of how transcripts for each individual HMTase change from the GV oocyte to the BL embryo revealed no significant change in G9a transcript abundance. GLP transcripts decrease 7-fold at the 4C stage (GV v. 4C, P < 0.05). No difference in relative abundance of ESET transcripts was detectable at GV, MII, or BL-IVF stages; ESET was not detectable in 4C-IVF embryos. SuvH1 transcripts decreased 9-fold in BL-IVF (GV v. BL, P < 0.05); SuvH2 transcripts decreased 21-fold at the 4C-IVF and BL-IVF stages (GV v. 4C, BL, P < 0.05). The differences in transcript abundance of these genes indicate that their expression may change in order to regulate the transcription of genes important in early development.

198 KARYOPHERIN ALPHA EXPRESSION IN PORCINE OOCYTES AND EMBRYOS PRODUCED BY IN VITRO FERTILIZATION

2009 ◽  
Vol 21 (1) ◽  
pp. 197
Author(s):  
X. Wang ◽  
L. Magnani ◽  
R. Cabot
Keyword(s):  
Internal Control ◽  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Transcript Abundance ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Significance Level ◽  
Nuclear Localization Signals ◽  
Importin Α

Partitioning intracellular proteins between nuclear and cytoplasmic compartments is critically important for coordinating major cellular events involved in transcription and differentiation. Import of cytoplasmic proteins bearing classical nuclear localization signals (NLSs) into the nucleus is mediated by the importin α/β heterodimer. Importin α, also called karyopherin α (KPNA), serves to recognize the NLS-bearing cytoplasmic cargo. Six KPNA molecules have been characterized in human (KPNA1-6). Select KPNA molecules are known to be differently expressed in specific tissues; individual KPNA molecules also have specificity for unique NLS-bearing cargos. We hypothesized that transcripts for individual porcine KPNA molecules would be present at differing levels at specific stages of oocyte maturation and cleavage development, thereby reflecting the changing requirements for particular import pathways during this window of development. To test this hypothesis, we first identified the porcine orthologs of KPNA1-6. We also identified the open reading frame of a potentially novel KPNA, KPNA7. KPNA7 was highly represented in the porcine EST database from expressed sequence tags derived from oocytes and ovarian tissue. Transcript abundance of KPNA1-7 was determined in germinal vesicle (GV) and MII-stage (MII) porcine oocytes and 4-cell (4C) and blastocyst-stage (BL) porcine embryos using quantitative real-time PCR. mRNA was isolated from pools (50 200) of GV and MII oocytes and 4C and BL embryos produced by IVF. Transcripts for KPNA1-7 and YWHAG (internal control for transcript normalization) were amplified in duplicate across 3 to 5 replicates. Relative transcript abundance of these genes was measured using the comparative CT method; GV was taken as the calibrator stage. Data were analyzed using GLM procedures in SAS (SAS Institute Inc., Cary, NC, USA) with the significance level at 0.05, and differences were compared by Tukey’s post test. Our results showed that KPNA1 had a significant decrease in MII oocytes (4-fold, GV v. MII). Transcript abundance of KPNA2 was significantly higher in GV oocytes and 4C embryos than in MII oocytes (2-fold GV v. MII; 3-fold 4C v. MII); KPNA2 transcripts were not detectable in BL embryos. KPNA3 transcripts were reduced in BL embryos compared to GV oocytes (8-fold, BL v. GV). KPNA4 transcripts were increased at the 4-cell stage (18-fold, GV v. 4C). The transcripts of KPNA5 were detectable only in GV and MII oocytes. No significant changes in the amount of KPNA6 transcripts were detectable at the stages analyzed. Transcript levels of KPNA7 were reduced in BL embryos as compared to the GV oocytes (1165-fold, BL v. GV). Throughout all these stages examined, KPNA5 had the lowest transcript abundance and was not detectable in 4C and BL stages. Transcripts levels of KPNA7 were in higher abundance than KPNA1-6 in GV and MII oocytes. Results suggest that KPNA7 is a new member of the KPNA family. Our results also suggest that porcine oocytes and embryos, at discrete stages of development, have differing requirements for individual KPNA molecules.

scholarly journals Perinatal Risks Associated with Early Vanishing Twin Syndrome following Transfer of Cleavage- or Blastocyst-Stage Embryos

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Nigel Pereira ◽  
Katherine P. Pryor ◽  
Allison C. Petrini ◽  
Jovana P. Lekovich ◽  
Jaclyn Stahl ◽  
...  
Keyword(s):  
Birth Weight ◽  
Low Birth Weight ◽  
Very Low Birth Weight ◽  
Blastocyst Stage ◽  
Cleavage Stage ◽  
Vitro Fertilization ◽  
Singleton Birth ◽  
Stage Embryo ◽  
Perinatal Risks

Objective. To investigate whether the perinatal risks associated with early vanishing twin (VT) syndrome differ between cleavage- or blastocyst-stage embryo transfers (ET) in fresh in vitro fertilization (IVF) cycles.Methods. Retrospective, single-center, cohort study of IVF cycles with fresh cleavage- or blastocyst-stage ETs resulting in a live singleton birth. The incidence of preterm birth (PTB), low birth weight (LBW), and very low birth weight (VLBW) was compared between cleavage- and blastocyst-stage ET cycles complicated by early VT.Results. 7241 patients had live singleton births. Early VT was observed in 709/6134 (11.6%) and 70/1107 (6.32%) patients undergoing cleavage-stage and blastocyst-stage ETs, respectively. Patients in the blastocyst-stage group were younger compared to the cleavage-stage group. The cleavage-stage group had a similar birth weight compared to the blastocyst-stage group. There was no difference in the incidence of PTB (9.87% versus 8.57%), LBW (11.1% versus 11.4%), or VLBW (1.13 versus 1.43%) when comparing the cleavage-stage early VT and blastocyst-stage early VT groups, even after adjustment with logistic regression.Conclusions. Our study highlights that the adverse perinatal risks of PTB, LBW, and VLBW associated with early VT syndrome are similar in patients undergoing cleavage-stage or blastocyst-stage ETs during fresh IVF cycles.

144 EPIGENETIC ASYMMETRY IN HISTONE H3 LYSINE 9 DIMETHYLATION STATUS IN PRONUCLEAR STAGE PORCINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 180
Author(s):  
M. Sega ◽  
R. Cabot
Keyword(s):  
In Vitro Fertilization ◽  
Histone H3 ◽  
Germinal Vesicle ◽  
Immunocytochemical Staining ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
H3k9 Dimethylation ◽  
Chi Square Analysis

Covalent modification of specific residues on the tail regions of core histone proteins has been shown to play a key role in regulation of the genome. Methylation of lysine 9 on histone H3 (H3K9) is associated with repression of transcription and formation of heterochromatin domains. The maternal- and paternal-derived pronuclei from pronuclear stage murine embryos have an asymmetric distribution of H3K9 dimethylation between the 2 pronuclei; maternal pronuclei have the H3K9 dimethylation, whereas the paternal pronuclei lack this modification. The aim of this study was to characterize the H3K9 dimethylation pattern in cleavage stage porcine embryos. Indirect immunocytochemical staining was performed using a commercially available antibody from Upstate (Charlottesville, VA) that recognizes the dimethylated form of lysine 9 on histone H3 and fluorescein isothiocyanate (FITC)-conjugated secondary antibody on germinal vesicle (GV) stage porcine oocytes and cleavage stage porcine embryos. Germinal vesicle stage oocytes were matured in vitro for 44 h in a chemically defined maturation medium (TCM 199 supplemented with 0.1% PVA, 0.069 mg/mL cysteine, 10 ng/mL EGF 0.5 IU/mL LH, and 0.5 IU/mL FSH) at 39�C in 5% CO2. Following in vitro fertilization, presumptive zygotes were cultured in NCSU23 medium containing 4 mg/mL BSA at 39�C in 5% CO2, 5% O2. Embryos were fixed in 3.7% paraformaldehide for 2 h and washed in PBS and 0.1% Tween20. H3K9 dimethylation is present in the nuclei of GV-stage oocytes (n = 24) and pronuclear (n = 13), 2-cell (n = 4), 4-cell (n = 9), blastocyst (n = 8) stage embryos. The analysis revealed 2 interesting findings. First, examination of thin optical sections through the nuclei of processed embryos on a confocal microscope revealed that in GV-stage oocytes and pronuclear, 2-cell and 4-cell stage embryos the dimethylated form of H3K9 was distributed throughout the nuclei at these developmental time points, whereas in blastocyst stage embryos, the dimethylated H3K9 was restricted to the nuclear periphery. Second, not all pronuclei within pronuclear stage embryos were positive for the dimethylated form of H3K9. To determine if differential H3K9 methylation pattern observed among pronuclei in 1-cell stage embryos was due to parental origin, we produced parthenogenic porcine embryos by electrically activating porcine oocytes in calcium containing activation medium and processed them as indicated above for fertilized oocytes (n = 13). All pronuclei found in pronuclear stage parthenogenic porcine embryos were positive for dimethylation of H3K9. Chi square analysis revealed this pattern to be different from that observed in pronuclear embryos produced by fertilization (P < 0.05). In summary we conclude that the pronuclei in 1-celled embryos produced by in vitro fertilization are differentially dimethylated at H3K9 and that the localization of dimethylated H3K9 changes during cleavage development.

scholarly journals Clinical Pregnancy after Elimination of Embryo Fragments Before Fresh Cleavage-Stage Embryo Transfer

2020 ◽  
Author(s):  
Luis H. Sordia-Hernandez ◽  
Felipe A. Morales-Martinez ◽  
Lorna M. Frazer-Moreira ◽  
Lilith Villarreal-Pineda ◽  
María Ofelia Sordia-Piñeyro ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Poor Quality ◽  
Abortion Rate ◽  
Pregnancy Rates ◽  
Clinical Pregnancy ◽  
Cleavage Stage ◽  
Fresh Embryo Transfer ◽  
Vitro Fertilization ◽  
Stage Embryo

Objective: To determine if the elimination of fragments in cleavage-stage embryos, before fresh transfer, improves pregnancy rates in in vitro fertilization cycles. Materials and methods: This is a Prospective observational case-control study carried out at a University Reproductive Center. We included Twenty-six infertile patients divided into two groups. Group one: 13 patients with embryos classified as grade B and C (embryos with fragments) according to the Hill classification, and Group two: 13 patients with grade A embryos (embryos with no fragments). Embryo Defragmentation was performed in embryos of group one 65 to 68 hours after conventional fertilization. Fresh embryo transfer was made after two hours post fragments removal. Reproductive results were evaluated and compared between both groups. Results: The total number of clinical pregnancies was nine. In group one there were 5 (38.5 %); in group two, there were 4 (30.8%). The difference was not statistically significant (p = 0.68). Two abortions were reported in the study, both in group one; were fragment elimination was performed. This represents an abortion rate of 40% in patients who got pregnant in this group. These patients had twice the probability of suffering an abortion (OR 2.1; 95% CI 1.4-3.37). Ongoing pregnancies were similar in both groups. Conclusion: Removal of fragments in freshly transferred day three embryos could be an alternative to increase clinical pregnancy and ongoing pregnancy rates in patients who have only poor-quality embryos. Despite the relationship with a higher abortion rate, this strategy could represent a real alternative for this type of patient.

Sign in / Sign up

Export Citation Format

Share Document